Recombinant rift valley fever (rvf) viruses and methods of use

ABSTRACT

Described herein are recombinant RVF viruses comprising deletions in one or more viral virulence genes, such as NSs and NSm. The recombinant RVF viruses, generated using a plasmid-based reverse genetics system, can be used as vaccines to prevent infection of RVF virus in livestock and humans. As described herein, the recombinant RVF viruses grow to high titers, provide protective immunity following a single injection and allow for the differentiation between vaccinated animals and animals infected with wild-type RVF virus.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. application Ser. No. 14/163,058, filed Jan.24, 2014, which is a divisional of U.S. application Ser. No. 12/809,561,filed Jun. 18, 2010, issued as U.S. Pat. No. 8,673,629, on Mar. 18,2014, which is the U.S. National Stage of International Application No.PCT/US2008/087023, filed Dec. 16, 2008, published in English under PCTArticle 21(2), which claims the benefit of U.S. Provisional ApplicationNo. 61/016,065, filed Dec. 21, 2007, and U.S. Provisional ApplicationNo. 61/042,987, filed Apr. 7, 2008. All of the above-listed applicationsare herein incorporated by reference in their entirety.

FIELD

This disclosure concerns recombinant RVF viruses comprising deletions inviral virulence genes. These recombinant viruses can be used as vaccinesto prevent RVF virus infection. This disclosure further relates to areverse genetics system used to generate recombinant RVF viruses.

BACKGROUND

Rift Valley fever (RVF) virus (family Bunyaviridae, genus Phlebovirus)is a mosquito-borne pathogen of both livestock and humans foundthroughout Africa and more recently the Arabian Peninsula. Historically,RVF virus has been the cause of either low-level endemic activity orlarge explosive epizootics/epidemics of severe disease throughout itsrange (Findlay et al., Lancet ii:1350-1351, 1931; Jouan et al., Res.Virol. 140:175-186, 1989; Ringot et al., Emerg. Infect. Dis. 10:945-947,2004; Woods et al., Emerg. Infect. Dis. 8:138-144, 2002). RVF outbreaksare characterized by economically disastrous “abortion storms” withnewborn animal mortality approaching 100% among livestock, especiallysheep and cattle (Coetzer et al., J. Vet. Res. 49:11-17, 1982; Easterdayet al., Am. J. Vet. Res. 23:470-479, 1962; Rippy et al., Vet. Pathol.29:495-502, 1992).

Human infections typically occur either from an infected mosquito bite,percutaneous/aerosol exposure during the slaughter of infected animals,or via contact with aborted fetal materials. Human RVF disease isprimarily a self-limiting febrile illness that in a small percentage(about 1-2%) of cases can progress to more serious and potentiallylethal complications including hepatitis, delayed onset encephalitis,retinitis, blindness, or a hemorrhagic syndrome with a hospitalized casefatality of 10-20% (Madani et al., Clin. Infect. Dis. 37:1084-1092,2003; McIntosh et al., S. Afr. Med. J. 58:03-806, 1980). Excessivelyheavy rainfall in semi-arid regions often precedes large periodicoutbreaks of RVF virus activity, allowing for the abundant emergence oftransovarially infected Aedes spp. mosquitoes and subsequent initiationof an outbreak by transmission of virus to livestock and humans viainfected mosquito feeding (Linthicum et al., Science 285:397-400, 1999;Swanepoel et al., Contributions to Epidemiology and Biostatistics3:83-91, 1981). The association with abnormally heavy rains providessome ability to predict periods and regions of high disease risk, whichin turn provides a potential window of opportunity for targetedvaccination programs if a safe, inexpensive and highly efficacioussingle dose vaccine were available.

The ability of RVF virus to cross international and geographicboundaries and strain veterinary and public health infrastructures iswell documented. In 1977, RVF virus was reported for the first timenorth of the Sahara desert where an extremely large outbreak affectingmore than 200,000 people occurred along the Nile River basin in Egypt(Meegan et al., Contributions to Epidemiology and Biostatistics3:100-113, 1981). Approximately ten years later in 1987, a largeoutbreak occurred in western Africa along the border of Mauritania andSenegal affecting an estimated 89,000 individuals (Jouan et al., Res.Virol. 140:175-186, 1989). Later, the virus was isolated for the firsttime outside of Africa (across the Red sea) in Saudi Arabia and Yemenand was found to be the cause of a large epizootic/epidemic in 2000 withan estimated 2000 human cases and 245 deaths (Anonymous, Morb. Mortal.Wkly. Rep. 49:982-5, 2000; Centers for Disease Control and Prevention,Morb. Mortal. Wkly. Rep. 49:1065-1066, 2000; Shoemaker et al., Emerg.Infect. Dis. 8:1415-1420, 2002).

Most recently, in late 2006 to early 2007, following heavy rainfall ineastern Africa, RVF virus emerged as the cause of a widespread outbreakthat eventually resulted in 1062 reported human cases and 315 reporteddeaths. Associated with the outbreak were substantial economic lossesamong livestock in southern Somalia, Kenya, and northern Tanzania(Anonymous, Morb. Mortal. Wkly. Rep. 56:73-76, 2007). The ability of RVFvirus to cause explosive outbreaks in previously unaffected regionsaccompanied by high morbidity and mortality during RVFepizootics/epidemics highlights the importance of developing highthroughput screening tools for potential antiviral therapeutic agentsand the development of safe and efficacious vaccines for thissignificant veterinary and public health threat.

SUMMARY

Disclosed are RVF viruses that are highly attenuated, immunogenic andcontain precise molecular markers allowing for the differentiation ofnaturally infected and vaccinated animals (DIVA). Provided herein arerecombinant RVF viruses, wherein the genome of the recombinant RVFviruses comprise (i) a full-length L segment; (ii) a full-length Msegment or an M segment comprising a complete deletion of the NSm openreading frame (ORF); and (iii) an S segment comprising a completedeletion of the NSs ORF. In one embodiment, the NSs ORF of therecombinant RVF virus is replaced by the ORF of a reporter gene.

Also provided are immunogenic compositions comprising one or more of therecombinant RVF viruses described herein and a pharmaceuticallyacceptable carrier. Further provided is a method of immunizing a subjectagainst RVF virus infection, comprising administering to the subject animmunogenic composition disclosed herein. The immunogenic compositionscan be used for vaccination of livestock or humans.

Further provided is a collection of plasmids comprising (i) a plasmidencoding a full-length anti-genomic copy of the L segment of RVF virus;(ii) a plasmid encoding a full-length anti-genomic copy of the M segmentof RVF virus, or an anti-genomic copy of the M segment of RVF viruscomprising a complete deletion of the NSm ORF; and (iii) a plasmidencoding an anti-genomic copy of the S segment of RVF virus, wherein theS segment comprises a complete deletion of the NSs ORF. In someembodiments, the plasmids further comprise a T7 promoter and a hepatitisdelta virus ribozyme. In one example, the NSs ORF of the S segmentplasmid is replaced by the ORF of a reporter gene, such as a greenfluorescent protein. Also provided are isolated host cells comprising acollection of plasmids provided herein.

Further provided is a method of preparing a recombinant RVF virus forimmunization of a subject, comprising (i) transfecting cultured cellswith the collection of plasmids described herein; (ii) incubating thecells for 1 to 5 days; and (iii) collecting recombinant RVF virus fromthe cell supernatant.

Also provided are recombinant RVF viruses, wherein the genome of therecombinant RVF viruses comprise a full-length L segment, a full-lengthM segment and a full-length S segment, wherein the S segment furtherencodes the ORF of a reporter gene.

A reverse genetics system for producing recombinant RVF virus is alsoprovided. The reverse genetics system consists of three plasmids, aplasmid that encodes an anti-genomic copy of an S segment, a plasmidthat encodes an M segment and a plasmid that encodes an L segment of RVFvirus, wherein each plasmid comprises a T7 promoter and a hepatitisdelta virus ribozyme. Further provided are recombinant RVF virusesproduced using the reverse genetics system described herein.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a schematic depiction of the rRVF cDNA plasmid constructionsused to generate the rRVF virus stocks. FIG. 1B is a series of imagesshowing direct live cell UV imaging of Vero E6 cells 24 hours afterinfection with each recombinant rRVF virus (expressing eGFP). FIG. 1C isa series of images showing Vero E6 cells 24 hours after infection withrRVF-NSs-(Ala)₃GFP or rRVF-NSs(Ala)₁₀GFP viruses at an MOI of 1. Cellswere fixed and stained with monoclonal antibodies specific for eGFP, RVFNSs and counterstained with DAPI to confirm intranuclear co-localizationof eGFP and NSs.

FIG. 2 is a graph showing the results of anti-RVF virus total IgGadjusted SUM_(OD) ELISA testing of all vaccinated (40) and shaminoculated (5) control animals at day 26 post-immunization. Apositive/negative cut-off value was established as the mean+3 standarddeviations of the sham inoculated SUM_(OD) values (open circles-dashedline). §=Significant differences between vaccinated and control groups(p-value <0.05).

FIGS. 3A-3D are a series of images showing representative results ofindirect fluorescent testing of serum collected from (FIG. 3A) WF ratssurviving challenge with RVF virus; (FIG. 3B) vaccinated WF rats (day 26post-vaccination); (FIG. 3C) negative control sham inoculated rats (day26 post-vaccination); and (FIG. 3D) naturally infected convalescentlivestock (goat) sera obtained during the RVF virus outbreak in SaudiArabia in 2000. The presence of anti-NP antibodies (left panels) andanti-NSs antibodies (right panels) is detected using Vero E6 cellsexpressing either NP or NSs, respectively. To confirm intranuclearaccumulation of anti-NSs antibody, cells were counterstained with DAPI.

FIGS. 4A and 4B are tables showing data obtained from a vaccinationpilot study (FIG. 4A) and challenge study (FIG. 4B).

FIG. 5 is a plasmid map of pRVS.

FIG. 6 is a plasmid map of pRVM.

FIG. 7 is a plasmid map of pRVL.

FIG. 8 is a plasmid map of pRVS-GFPΔNSs.

FIG. 9 is a plasmid map of pRVMΔNSm.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. The Sequence Listing is submitted as an ASCII textfiled, created on Aug. 1, 2016, 59 KB. In the accompanying sequencelisting:

SEQ ID NO: 1 is the nucleotide sequence of the S segment of wild-typeRVF virus strain ZH501, deposited under GenBank Accession No. DQ380149on Jan. 31, 2007.

SEQ ID NO: 2 is the nucleotide sequence of the M segment of wild-typeRVF virus strain ZH501, deposited under GenBank Accession No. DQ380200on Jan. 31, 2007.

SEQ ID NO: 3 is the nucleotide sequence of the L segment of wild-typeRVF virus strain ZH501, deposited under GenBank Accession No. DQ375406on Jan. 31, 2007.

SEQ ID NO: 4 is the nucleotide sequence of primer RVS-35/KpnI.

SEQ ID NO: 5 is the nucleotide sequence of primer RVS+827/BglII.

SEQ ID NO: 6 is the nucleotide sequence of primer eGFP+1/KpnI.

SEQ ID NO: 7 is the nucleotide sequence of primer eGFP-720/BglII.

SEQ ID NO: 8 is the nucleotide sequence of primer RVS-829rev/KpnI.

SEQ ID NO: 9 is the nucleotide sequence of primer NSsGFP+10Ala/Fwd.

SEQ ID NO: 10 is the nucleotide sequence of primer NSsGFP+10Ala/Rev.

SEQ ID NO: 11 is the nucleotide sequence of plasmid pRVS.

SEQ ID NO: 12 is the nucleotide sequence of plasmid pRVM.

SEQ ID NO: 13 is the nucleotide sequence of plasmid pRVL.

SEQ ID NO: 14 is the nucleotide sequence of plasmid pRVS-GFPΔNSs.

SEQ ID NO: 15 is the nucleotide sequence of the plasmid pRVMΔNSm.

DETAILED DESCRIPTION I. Abbreviations

-   -   BSL Bio-safety level    -   CPE Cytopathic effect    -   DIVA Differentiation of naturally infected and vaccinated        animals    -   eGFP Enhanced green fluorescent protein    -   HRP Horseradish peroxidase    -   IFA Immunofluorescence assay    -   LD Lethal dose    -   MOI Multiplicity of infection    -   NP Nucleoprotein    -   NS Non-structural    -   OIE Office International des Epizooties    -   ORF Open reading frame    -   PFU Plaque-forming unit    -   PRNT Plaque reduction neutralization titers    -   RNA Ribonucleic acid    -   RT-PCR Reverse transcriptase polymerase chain reaction    -   RVF Rift Valley fever    -   SQ Subcutaneously    -   USDA United States Department of Agriculture    -   WF Wistar-furth    -   WOAH World Organization for Animal Health

II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

Adjuvant: A substance or vehicle that non-specifically enhances theimmune response to an antigen. Adjuvants can include a suspension ofminerals (alum, aluminum hydroxide, or phosphate) on which antigen isadsorbed; or water-in-oil emulsion in which antigen solution isemulsified in mineral oil (for example, Freund's incomplete adjuvant),sometimes with the inclusion of killed mycobacteria (Freund's completeadjuvant) to further enhance antigenicity. Immunostimulatoryoligonucleotides (such as those including a CpG motif) can also be usedas adjuvants (for example, see U.S. Pat. Nos. 6,194,388; 6,207,646;6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705; and 6,429,199).Adjuvants also include biological molecules, such as costimulatorymolecules. Exemplary biological adjuvants include IL-2, RANTES, GM-CSF,TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L and 41 BBL.

Administer: As used herein, administering a composition to a subjectmeans to give, apply or bring the composition into contact with thesubject. Administration can be accomplished by any of a number ofroutes, such as, for example, topical, oral, subcutaneous,intramuscular, intraperitoneal, intravenous, intrathecal andintramuscular.

Ambisense: Refers to a genome or genomic segments having both positivesense and negative sense portions. For example, the S segment of aPhlebovirus, such as Rift Valley fever virus, is ambisense, encodingnucleoprotein (NP) in the negative sense and the non-structural protein(NSs) in the positive sense.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and bird. Mammals include, but are notlimited to, humans, non-human primates, dogs, cats, horses, sheep andcows. The term mammal includes both human and non-human mammals.

Antibody: An immunoglobulin molecule produced by B lymphoid cells with aspecific amino acid sequence. Antibodies are evoked in humans or otheranimals by a specific antigen (immunogen). Antibodies are characterizedby reacting specifically with the antigen in some demonstrable way,antibody and antigen each being defined in terms of the other.“Eliciting an antibody response” refers to the ability of an antigen orother molecule to induce the production of antibodies.

Antigen: A compound, composition, or substance that can stimulate theproduction of antibodies or a T-cell response in an animal, includingcompositions that are injected or absorbed into an animal. An antigenreacts with the products of specific humoral or cellular immunity,including those induced by heterologous immunogens. In one embodiment,an antigen is a RVF virus antigen.

Anti-genomic: As used herein, “anti-genomic” refers to a genomic segmentof a virus that encodes a protein in the orientation opposite to theviral genome. For example, Rift valley fever virus is a negative-senseRNA virus. However, the S segment is ambisense, encoding proteins inboth the positive-sense and negative-sense orientations. “Anti-genomic”refers to the positive-sense orientation, while “genomic” refers to thenegative-sense orientation.

Attenuated: In the context of a live virus, the virus is attenuated ifits ability to infect a cell or subject and/or its ability to producedisease is reduced (for example, eliminated) compared to a wild-typevirus. Typically, an attenuated virus retains at least some capacity toelicit an immune response following administration to an immunocompetentsubject. In some cases, an attenuated virus is capable of eliciting aprotective immune response without causing any signs or symptoms ofinfection. In some embodiments, the ability of an attenuated virus tocause disease in a subject is reduced at least about 10%, at least about25%, at least about 50%, at least about 75% or at least about 90%relative to wild-type virus.

Fusion protein: A protein generated by expression of a nucleic acidsequence engineered from nucleic acid sequences encoding at least aportion of two different (heterologous) proteins. To create a fusionprotein, the nucleic acid sequences must be in the same reading frameand contain to internal stop codons.

Hepatitis delta virus ribozyme: A non-coding, catalytic RNA from thehepatitis delta virus. Ribozymes catalyze the hydrolysis of their ownphosphodiester bonds or those of other RNA molecules.

Host cell: In the context of the present disclosure, a “host cell” is acell of use with the RVF virus reverse genetics systems describedherein. A suitable host cell is one that is capable of transfection withand expression of the plasmids of the RVF virus reverse genetics system.In one embodiment, the host cell is a cell expressing the T7 polymerase,such as, but not limited to BSR-T7/5 cells (Buchholz et al., J. Virol.73(1):251-259, 1999).

Immune response: A response of a cell of the immune system, such as aB-cell, T-cell, macrophage or polymorphonucleocyte, to a stimulus suchas an antigen. An immune response can include any cell of the bodyinvolved in a host defense response, including for example, anepithelial cell that secretes an interferon or a cytokine. An immuneresponse includes, but is not limited to, an innate immune response orinflammation. As used herein, a protective immune response refers to animmune response that protects a subject from infection (preventsinfection or prevents the development of disease associated withinfection).

Immunogen: A compound, composition, or substance which is capable, underappropriate conditions, of stimulating an immune response, such as theproduction of antibodies or a T-cell response in an animal, includingcompositions that are injected or absorbed into an animal. As usedherein, as “immunogenic composition” is a composition comprising animmunogen.

Immunize: To render a subject protected from an infectious disease, suchas by vaccination.

Isolated: An “isolated” biological component (such as a nucleic acid,protein or virus) has been substantially separated or purified away fromother biological components (such as cell debris, or other proteins ornucleic acids). Biological components that have been “isolated” includethose components purified by standard purification methods. The termalso embraces recombinant nucleic acids, proteins or viruses, as well aschemically synthesized nucleic acids or peptides.

Linker: One or more amino acids that serve as a spacer between twopolypeptides of a fusion protein.

Livestock: Domesticated animals reared in an agricultural setting as asource of food or to provide labor. The term “livestock” includes, butis not limited to, cattle, deer, donkeys, goats, horses, mules, rabbitsand sheep.

ORF (open reading frame): A series of nucleotide triplets (codons)coding for amino acids without any termination codons. These sequencesare usually translatable into a peptide.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein-coding regions, in the samereading frame.

Pharmaceutically acceptable vehicles: The pharmaceutically acceptablecarriers (vehicles) useful in this disclosure are conventional.Remington's Pharmaceutical Sciences, by E. W. Martin, Mack PublishingCo., Easton, Pa., 15th Edition (1975), describes compositions andformulations suitable for pharmaceutical delivery of one or moretherapeutic compounds or molecules, such as one or more recombinant RVFviruses, and additional pharmaceutical agents.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (for example, powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically-neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Plasmid: A circular nucleic acid molecule capable of autonomousreplication in a host cell.

Polypeptide: A polymer in which the monomers are amino acid residueswhich are joined together through amide bonds. When the amino acids arealpha-amino acids, either the L-optical isomer or the D-optical isomercan be used. The terms “polypeptide” or “protein” as used herein areintended to encompass any amino acid sequence and include modifiedsequences such as glycoproteins. The term “polypeptide” is specificallyintended to cover naturally occurring proteins, as well as those whichare recombinantly or synthetically produced. The term “residue” or“amino acid residue” includes reference to an amino acid that isincorporated into a protein, polypeptide, or peptide.

Conservative amino acid substitutions are those substitutions that, whenmade, least interfere with the properties of the original protein, thatis, the structure and especially the function of the protein isconserved and not significantly changed by such substitutions. Examplesof conservative substitutions are shown below.

Original Residue Conservative Substitutions Ala Ser Arg Lys Asn Gln, HisAsp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; ValLys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp TyrTyr Trp; Phe Val Ile; Leu

Conservative substitutions generally maintain (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.

The substitutions which in general are expected to produce the greatestchanges in protein properties will be non-conservative, for instancechanges in which (a) a hydrophilic residue, for example, seryl orthreonyl, is substituted for (or by) a hydrophobic residue, for example,leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, for example, lysyl, arginyl, orhistadyl, is substituted for (or by) an electronegative residue, forexample, glutamyl or aspartyl; or (d) a residue having a bulky sidechain, for example, phenylalanine, is substituted for (or by) one nothaving a side chain, for example, glycine.

Preventing, treating or ameliorating a disease: “Preventing” a diseaserefers to inhibiting the full development of a disease. “Treating”refers to a therapeutic intervention that ameliorates a sign or symptomof a disease or pathological condition after it has begun to develop.“Ameliorating” refers to the reduction in the number or severity ofsigns or symptoms of a disease.

Promoter: A promoter is an array of nucleic acid control sequences whichdirect transcription of a nucleic acid. A promoter includes necessarynucleic acid sequences near the start site of transcription. A promoteralso optionally includes distal enhancer or repressor elements. A“constitutive promoter” is a promoter that is continuously active and isnot subject to regulation by external signals or molecules. In contrast,the activity of an “inducible promoter” is regulated by an externalsignal or molecule (for example, a transcription factor). In oneembodiment, the promoter is a T7 promoter (from bacteriophage T7).

Purified: The term “purified” does not require absolute purity; rather,it is intended as a relative term. Thus, for example, a purifiedpeptide, protein, virus, or other active compound is one that isisolated in whole or in part from naturally associated proteins andother contaminants. In certain embodiments, the term “substantiallypurified” refers to a peptide, protein, virus or other active compoundthat has been isolated from a cell, cell culture medium, or other crudepreparation and subjected to fractionation to remove various componentsof the initial preparation, such as proteins, cellular debris, and othercomponents.

Recombinant: A recombinant nucleic acid, protein or virus is one thathas a sequence that is not naturally occurring or has a sequence that ismade by an artificial combination of two otherwise separated segments ofsequence. This artificial combination is often accomplished by chemicalsynthesis or, more commonly, by the artificial manipulation of isolatedsegments of nucleic acids, for example, by genetic engineeringtechniques. In one embodiment, recombinant RVF virus is generated usingthe reverse genetics system described herein. In some examples, therecombinant RVF viruses comprise one or more deletions in a viralvirulence factor, such as NSs and/or NSm. In other examples, therecombinant RVF viruses comprise a heterologous gene, such as a reportergene.

Reporter gene: A reporter gene is a gene operably linked to another geneor nucleic acid sequence of interest (such as a promoter sequence).Reporter genes are used to determine whether the gene or nucleic acid ofinterest is expressed in a cell or has been activated in a cell.Reporter genes typically have easily identifiable characteristics, suchas fluorescence, or easily assayed products, such as an enzyme. Reportergenes can also confer antibiotic resistance to a host cell or tissue.Reporter genes include, for example, GFP (or eGFP) or other fluorescencegenes, luciferase, β-galactosidase and alkaline phosphatase.

Reverse genetics: Refers to the process of introducing mutations (suchas deletions, insertions or point mutations) into the genome of anorganism or virus in order to determine the phenotypic effect of themutation. For example, introduction of a mutation in a specific viralgene enables one to determine the function of the gene.

Rift Valley fever (RVF) virus: A virus belonging to the familyBunyaviridae and genus Phlebovirus. RVF virus has a single-stranded,negative-sense genome composed of three genome segments, S, M and L. TheS segment is an ambisense genome segment, meaning it encodes proteins inboth the positive-sense and negative-sense orientations. The RVF virusgenome encodes both structural and non-structural proteins. A“structural” protein is a protein found in the virus particle, whereas a“non-structural” protein is only expressed in a virus-infected cell. RVFvirus structural proteins include nucleoprotein (NP or N, usedinterchangeably), two glycoproteins (Gn and Gc) and the viralRNA-dependent RNA polymerase (L protein). Non-structural RVF virusproteins include NSs, NSm and the NSm+Gn fusion protein. As used herein,a “full-length” RVF virus genome segment is one containing no deletions.Full-length genome segments can contain mutations or substitutions, butretain the same length as the wild-type virus. A “complete deletion” ofan ORF of a RVF virus genome segment means either every nucleotideencoding the ORF is deleted from genome segment, or nearly everynucleotide encoding the ORF is deleted such that no proteins aretranslated from the ORF. Thus, a “complete deletion” includes genomesegments retaining up to ten nucleotides encoding the ORF, such as one,two, three, four, five, six, seven, eight, nine or ten nucleotides. Anumber of RVF virus strains have been identified. In one embodimentdescribed herein, the RVF virus strain is ZH501.

As used herein, plasmids encoding full-length RVF virus S, M and Lgenome segments are referred to as pRVS, pRVM, and pRVL, respectively.The S segment plasmid containing the eGFP ORF in place of the completeNSs ORF is referred to as pRVS-GFPΔNSs. The M segment plasmid containinga complete deletion of the NSm ORF is referred to as pRVM-ΔNSm. Therecombinant RVF viruses based upon the ZH501 genome that are generatedusing reverse genetics are referred to as either rRVF or rZH501. Forexample, recombinant RVF virus generated using the pRVS-GFPΔNSs plasmid(and wild-type M and L plasmids), is referred to as rRVF-ΔNSs:GFP orrZH501-ΔNSs:GFP. Similarly, recombinant RVF virus generated using thepRVS-GFPΔNSs plasmid and pRVM-ΔNSm plasmid (and wild-type L plasmid),referred to as rRVF-ΔNSs:GFP-ΔNSm or rZH501-ΔNSs:GFP-ΔNSm. RecombinantRVF virus comprising wild-type M and L segments, and an S segmentencoding an NSs-eGFP fusion protein with a three alanine residue linker,is referred to as rRVF-NSs(Ala)₃GFP or rZH501-NSs(Ala)₃GFP). If thefusion protein comprises a ten alanine residue linker, the recombinantvirus is referred to as rRVF-NSs(Ala)₁₀GFP or rZH501-NSs(Ala)₁₀GFP.

Sequence identity: The similarity between amino acid or nucleic acidsequences is expressed in terms of the similarity between the sequences,otherwise referred to as sequence identity. Sequence identity isfrequently measured in terms of percentage identity (or similarity orhomology); the higher the percentage, the more similar the two sequencesare. Homologs or variants of a given gene or protein will possess arelatively high degree of sequence identity when aligned using standardmethods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J.Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci.U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237-244, 1988; Higginsand Sharp, CABIOS 5:151-153, 1989; Corpet et al., Nucleic Acids Research16:10881-10890, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci.U.S.A. 85:2444, 1988. Altschul et al., Nature Genet. 6:119-129, 1994.

The NCBI Basic Local Alignment Search Tool (BLAST™) (Altschul et al., J.Mol. Biol. 215:403-410, 1990) is available from several sources,including the National Center for Biotechnology Information (NCBI,Bethesda, Md.) and on the Internet, for use in connection with thesequence analysis programs blastp, blastn, blastx, tblastn and tblastx.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes both human and non-human mammals. Subjects include veterinarysubjects, including livestock such as cows and sheep, and non-humanprimates.

Therapeutically effective amount: A quantity of a specified agentsufficient to achieve a desired effect in a subject being treated withthat agent. For example, this may be the amount of a recombinant RVFvirus useful for eliciting an immune response in a subject and/or forpreventing infection by RVF virus. Ideally, in the context of thepresent disclosure, a therapeutically effective amount of a recombinantRVF virus is an amount sufficient to increase resistance to, prevent,ameliorate, and/or treat infection caused by RVF virus in a subjectwithout causing a substantial cytotoxic effect in the subject. Theeffective amount of a recombinant RVF virus useful for increasingresistance to, preventing, ameliorating, and/or treating infection in asubject will be dependent on, for example, the subject being treated,the manner of administration of the therapeutic composition and otherfactors.

Transformed: A transformed cell is a cell into which has been introduceda nucleic acid molecule by molecular biology techniques. As used herein,the term transformation encompasses all techniques by which a nucleicacid molecule might be introduced into such a cell, includingtransfection with viral vectors, transformation with plasmid vectors,and introduction of naked DNA by electroporation, lipofection, andparticle gun acceleration.

Vaccine: A preparation of immunogenic material capable of stimulating animmune response, administered for the prevention, amelioration, ortreatment of infectious or other types of disease. The immunogenicmaterial may include attenuated or killed microorganisms (such asattenuated viruses), or antigenic proteins, peptides or DNA derived fromthem. Vaccines may elicit both prophylactic (preventative) andtherapeutic responses. Methods of administration vary according to thevaccine, but may include inoculation, ingestion, inhalation or otherforms of administration. Inoculations can be delivered by any of anumber of routes, including parenteral, such as intravenous,subcutaneous or intramuscular. Vaccines may be administered with anadjuvant to boost the immune response.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. Hence “comprisingA or B” means including A, or B, or A and B. It is further to beunderstood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including explanations of terms, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

III. Overview of Several Embodiments

Disclosed herein are RVF virus vaccine candidates that are highlyattenuated, immunogenic and contain precise molecular markers allowingfor the differentiation of naturally infected and vaccinated animals(DIVA). The recombinant RVF viruses described herein, which in someembodiments contain complete deletions of one or both of the major virusvirulence factors, NSs and NSm, are highly attenuated in vivo, inducerobust anti-RVF antibody responses, provide protection from virulentvirus challenge and allow for the assessment of vaccination status inanimals on the basis of NSs/NP serology. Further provided arerecombinant RVF viruses comprising a reporter moiety, such as eGFP,which have a variety of applications, including as live virus researchtools that can be useful for the rapid screening of antiviraltherapeutic compounds.

Provided herein are recombinant RVF viruses, wherein the genome of therecombinant RVF viruses comprise (i) a full-length L segment; (ii) afull-length M segment or an M segment comprising a complete deletion ofthe NSm open reading frame (ORF); and (iii) an S segment comprising acomplete deletion of the NSs ORF. In one embodiment, the NSs ORF of therecombinant RVF virus is replaced by the ORF of a reporter gene.Reporter genes include, but are not limited to genes encodingfluorescent proteins, enzymes or antibiotic resistance. Any gene thatproduces a protein with a functional readout can be used as the reportergene. In one example, the reporter gene is a green fluorescent protein(GFP), such as enhanced GFP (eGFP).

The genome of the recombinant RVF viruses provided herein can be derivedfrom any strain or variant of RVF virus. In some embodiments, the genomeis derived from ZH501, ZH548, SA75 or SPB 9800523. In a preferredembodiment, the genome is derived from RVF virus strain ZH501. Thesequences of the S, M and L segments of the ZH501 strain are depositedunder GenBank Accession Nos. DQ380149 (SEQ ID NO: 1), DQ380200 (SEQ IDNO: 2) and DQ375406 (SEQ ID NO: 3), respectively. The nucleotidesequences of the S, M and L segments need not be 100% identical to thesequences provided herein. In some examples, the S, M and L segments areat least 80%, at least 85%, at least 90%, at least 95%, or at least 99%identical to a known or disclosed RVF virus S, M or L segment, such asthe S, M and L segments set forth as SEQ ID NO: 1, SEQ ID NO: 2 and SEQID NO: 3, respectively.

Also provided are immunogenic compositions comprising the recombinantRVF viruses described herein and a pharmaceutically acceptable carrier.Suitable pharmaceutical carriers are described herein and are well knownin the art. The pharmaceutical carrier used depends on a variety offactors, including the route of administration. In one embodiment, theimmunogenic composition further comprises an adjuvant. The adjuvant canbe any substance that improves the immune response to the recombinantRVF virus.

Further provided is a method of immunizing a subject against RVF virusinfection, comprising administering to the subject an immunogeniccomposition disclosed herein. In one embodiment, the subject islivestock. Livestock includes, but is not limited to sheep and cattle.In another embodiment, the subject is a human. In one example, theimmunogenic composition is administered in a single dose. In anotherembodiment, the immunogenic composition is administered in multipledoses, such as two, three or four doses. When administered in multipledoses, the time period between doses can vary. In some cases, the timeperiod is days, weeks or months. The immunogenic composition can beadministered using any suitable route of administration. Generally, therecombinant RVF viruses are administered parenterally, such asintramuscularly, intravenously or subcutaneously.

Further provided is a collection of plasmids comprising (i) a plasmidencoding a full-length anti-genomic copy of the L segment of RVF virus;(ii) a plasmid encoding a full-length anti-genomic copy of the M segmentof RVF virus, or an anti-genomic copy of the M segment of RVF viruscomprising a complete deletion of the NSm ORF; and (iii) a plasmidencoding an anti-genomic copy of the S segment of RVF virus, wherein theS segment comprises a complete deletion of the NSs ORF. In someembodiments, the plasmids further comprise a T7 promoter and a hepatitisdelta virus ribozyme. In one example, the NSs ORF of the S segmentplasmid is replaced by the ORF of a reporter gene. In one example, thereporter gene is green fluorescent protein. In some embodiments, the RVFvirus is ZH501.

In one embodiment, the nucleotide sequence of the S segment plasmid isat least 95% identical to the nucleotide sequence of SEQ ID NO: 14. Inparticular examples, the nucleotide sequence of the S segment plasmidcomprises the nucleotide sequence of SEQ ID NO: 14. In anotherembodiment, the nucleotide sequence of the M segment plasmid is at least95% identical to the nucleotide sequence of SEQ ID NO: 12. In particularexamples, the nucleotide sequence of the M segment plasmid comprises thenucleotide sequence of SEQ ID NO: 12. In another embodiment, thenucleotide sequence of the M segment plasmid is at least 95% identicalto the nucleotide sequence of SEQ ID NO: 15. In particular examples, thenucleotide sequence of the M segment plasmid comprises the nucleotidesequence of SEQ ID NO: 15. In another embodiment, the nucleotidesequence of the L segment plasmid is at least 95% identical to thenucleotide sequence of SEQ ID NO: 13. In particular examples, thenucleotide sequence of the L segment plasmid comprises the nucleotidesequence of SEQ ID NO: 13.

Also provided are isolated host cells comprising the collection ofplasmids provided herein. In one embodiment, the cells express T7polymerase.

Further provided is a method of preparing a recombinant RVF virus forimmunization of a subject, comprising (i) transfecting cultured cellswith the collection of plasmids described herein; (ii) incubating thecells for 1 to 5 days; and (iii) collecting recombinant RVF virus fromthe cell supernatant. In one embodiment, the cells express T7polymerase.

Further provided are recombinant RVF viruses, wherein the genome of therecombinant RVF viruses comprise a full-length L segment, a full-lengthM segment and a full-length S segment, wherein the S segment furtherencodes the ORF of a reporter gene. In one embodiment, the S segmentencodes a NSs-reporter gene fusion protein. In one aspect, the reportergene is fused to the C-terminus of NSs. In some examples, the fusionprotein further comprises a linker. The linker can be any suitablecombination of one or more amino acids. In one embodiment, the linkercomprises 3 to 10 alanine residues. In another embodiment, the reportergene is GFP.

Also provided herein is a reverse genetics system for producingrecombinant RVF virus. The reverse genetics system consists of threeplasmids, wherein a first plasmid encodes an anti-genomic copy of a Ssegment, a second plasmid encodes an anti-genomic copy of a M segmentand a third plasmid encodes an anti-genomic copy of a L segment of RVFvirus, and wherein each plasmid comprises a T7 promoter and a hepatitisdelta virus ribozyme. In some embodiments, the RVF virus is ZH501. Insome embodiments, the S, M and L segments are wild-type S, M and Lsegments. In another embodiment, the S segment comprises a deletion ofthe NSs ORF. In another embodiment, the M segment comprises a deletionof the NSm ORF. In another embodiment, the S segment comprises adeletion of the NSs ORF and the M segment comprises a deletion of theNSm ORF.

In one embodiment, the nucleotide sequence of the S segment plasmid isat least 95% identical to the nucleotide sequence of SEQ ID NO: 11. Inparticular examples, the nucleotide sequence of the S segment plasmidcomprises the nucleotide sequence of SEQ ID NO: 11. In anotherembodiment, the nucleotide sequence of the S segment plasmid is at least95% identical to the nucleotide sequence of SEQ ID NO: 14. In particularexamples, the nucleotide sequence of the S segment plasmid comprises thenucleotide sequence of SEQ ID NO: 14. In another embodiment, thenucleotide sequence of the M segment plasmid is at least 95% identicalto the nucleotide sequence of SEQ ID NO: 12. In particular examples, thenucleotide sequence of the M segment plasmid comprises the nucleotidesequence of SEQ ID NO: 12. In another embodiment, the nucleotidesequence of the M segment plasmid is at least 95% identical to thenucleotide sequence of SEQ ID NO: 15. In particular examples, thenucleotide sequence of the M segment plasmid comprises the nucleotidesequence of SEQ ID NO: 15. In another embodiment, the nucleotidesequence of the L segment plasmid is at least 95% identical to thenucleotide sequence of SEQ ID NO: 13. In particular examples, thenucleotide sequence of the L segment plasmid comprises the nucleotidesequence of SEQ ID NO: 13.

Typically, the three plasmids are transfected simultaneously into cellsexpressing T7 polymerase. After a sufficient period of time to allow forproduction of recombinant virus, such as about 1 to about 5 days,recombinant RVF virus is collected from the cell supernatant.

Further provided are recombinant RVF viruses produced using the reversegenetics system described herein. In some examples, the recombinant RVFviruses comprise a deletion in one or more viral virulence factors. Inone embodiment, the recombinant viruses comprise a complete deletion ofthe NSs ORF. In another embodiment, the recombinant viruses comprise acomplete deletion of the NSm ORF. In yet another embodiment, therecombinant viruses comprise a deletion in both the NSs and NSm ORFs.The recombinant RVF viruses can be used for both research andtherapeutic purposes. For example, the recombinant RVF viruses describedherein are suitable for use as vaccines to prevent, treat or ameliorateRVF virus infection in livestock and humans.

IV. Rift Valley Fever Virus Genome and Encoded Proteins

Like other members of the genus Phlebovirus, RVF virus has anegative-sense, single-stranded tripartite RNA genome composed of the S,M and L segments (Schmaljohn, and Hooper, Bunyaviridae: The Viruses andtheir Replication In Fields' Virology, Lippincott Williams & Wilkins,Philadelphia, Pa., 2001). The small (S) segment (about 1.6 kB) encodes,in an ambisense fashion, the virus nucleoprotein (NP) in the genomic (−)orientation, and the non-structural (NSs) protein in the anti-genomic(+) orientation (Albarino et al., J. Virol. 81:5246-5256, 2007). Themedium (M) segment (about 3.8 kB) encodes a least four nested proteinsin a single ORF, including two structural glycoproteins, Gn and Gc, andtwo nonstructural proteins, the 14 kD NSm and a 78 kD NSm+Gn fusion(Gerrard et al., Virology 359:459-465, 2007; Gerrard and Nichol,Virology 357:124-133, 2007). The M segment contains five conservedin-frame AUG-methionine start codons within the NSm protein codingregion at anti-genomic sense positions 21, 135, 174, 411 and 426.Alternate utilization of the AUGs at positions 21 or 135 results inexpression of the 14 kDa NSm protein and the 78 kD NSm+Gn fusion protein(Suzich et al., J. Virol. 64:1549-1555, 1990, Gerrard and Nichol,Virology 357:124-133, 2007). The large (L) segment (about 6.4 kB)encodes the viral RNA-dependent RNA polymerase (L protein).

RVF virus NP and L proteins are required for viral RNA synthesis(Ikegami et al., J. Virol. 79:5606-5615, 2005; Lopez et al., J. Virol.69:3972-3979, 1995). Gn and Gc are believed to mediate binding to an asyet unidentified receptor. The 78 kD NSm+Gn fusion protein has beenreported to be dispensable for viral replication in cell culture (Won etal., J. Virol. 80:8274-8278, 2006).

Both nonstructural genes (NSs and NSm) have been reported to function asvirus virulence factors and determinants of mammalian host pathogenesis(Bird et al., Virology 362:10-15, 2007; Vialat et al., J. Virol.74:1538-1543, 2000; Won et al., J. Virol. 24:13335-13345, 2007). NSsmediates the pan-downregulation of mRNA production by inhibition of RNApolymerase II activity (Billecocq et al., J. Virol. 78:9798-9806, 2004;Le May et al., Cell 116:541-550, 2004). Via this mechanism, the NSsprotein performs a critical role in mammalian host pathogenesis byindirectly disrupting the host cell antiviral response (Bouloy et al.,J. Virol., 75:1371-1377, 2001; Muller et al., Am. J. Trop. Med. Hyg.53:405-411, 1995; Vialat et al., J. Virol. 74:1538-1543, 2000).

Studies of the non-structural gene located on the RVF virus M segment(NSm) indicate it is dispensable for efficient RVF virus growth in bothIFN-competent and IFN-deficient cell culture (Gerrard et al., Virology359:459-465, 2007; Won et al., J. Virol. 80:8274-8278, 2006). However,further work utilizing a highly sensitive animal model revealed thatrecombinant RVF virus lacking the entire NSm coding region (rRVF-ΔNSm)was attenuated yet retained the ability to cause either acute-onsetlethal hepatic necrosis or delayed-onset lethal neurologic disease in aminority (44%) of animals (Bird et al., Virology 362:10-15, 2007). Otherstudies have shown that NSm functions as a virus virulence factor bysuppressing the host cell apoptotic pathway following infection (Won etal., J. Virol. 24:13335-13345, 2007).

V. Reverse Genetics System for RVF Virus

The ability to generate recombinant viruses containing selectedmutations and/or deletions is a powerful tool for the development ofvirus vaccines. Reverse genetics systems have been described for a fewviruses of the Bunyaviridae family, including Bunyamvera virus (Bridgenand Elliott, Proc. Natl. Acad. Sci. USA 93:15400-15404, 1996) and LaCrosse virus (Blakqori and Weber, J. Virol. 79:10420-10428, 2005).Recently, a reverse genetics system for a vaccine strain (MP-12) of RVFvirus was reported (U.S. Pre-Grant Publication No. 2007/0122431, hereinincorporated by reference).

The recombinant RVF viruses described herein are generated using anoptimized reverse genetics system capable of rapidly generatingwild-type and mutant viruses (Bird et al., Virology 362:10-15, 2007;Gerrard et al., Virology 359:459-465, 2007, each of which is hereinincorporated by reference). The RVF virus reverse genetics system is aT7 RNA polymerase-driven plasmid-based genetic system based on thegenome of the virulent RVF virus Egyptian isolate ZH501. This system,described in detail in Bird et al. (Virology 362:10-15, 2007) and in theExamples below, includes three plasmids expressing anti-genomic copiesof the S, M and L segments of ZH501. As used herein, the plasmids arereferred to as the pRVS, pRVM and pRVL plasmids, respectively.

Rescue of recombinant viruses is accomplished by simultaneoustransfection of the three anti-genomic sense plasmids into cells stablyexpressing T7 polymerase (for example, BSR-T7/5 cells). The genomesegments of each plasmid are flanked by a T7 promoter, which enablesgeneration of the primary RNA transcript, and the hepatitis delta virusribozyme, which removes extraneous nucleotides from the 3′ end of theprimary transcriptional products. The T7 RNA polymerase generatedtranscripts are identical copies of the RVF virus genome segments, withthe exception of an extra G nucleotide on the 5′ end derived from the T7promoter. When expressed in transfected host cells, the pRVS, pRVM andpRVL plasmids generate anti-genomic sense copies of the S, M and Lsegments, respectively.

In one embodiment, the recombinant RVF viruses are generated using anS-segment plasmid that comprises a deletion in the NSs gene. In oneexample, the deletion is a deletion of the entire NSs ORF. In oneaspect, the NSs ORF is replaced by the eGFP ORF. In another embodiment,the recombinant RVF viruses are generated using an S-segment plasmidcomprising a deletion in the NSs gene and an M-segment plasmidcomprising a deletion in the NSm ORF. In one example, the deletion is adeletion of the entire NSm ORF. In another embodiment, the recombinantRVF viruses are generated using full-length S, M and L plasmids, whereinone of the plasmids further encodes eGFP. In one example, the eGFP ORFis encoded by the S plasmid as a NSs-eGFP fusion protein.

VI. Use of Recombinant RVF Viruses

Recombinant RVF viruses generated using the reverse genetics systemdescribed herein can be used for both research and therapeutic purposes.Using this system, recombinant RVF viruses can be generated that containprecisely defined deletions of major virus virulence factors on one ormore genome segments. For example, viruses can be produce that containdeletions of NSs and/or NSm. Accordingly, such recombinant RVF virusescan be used as vaccines to prevent infection of livestock and humanswith wild-type RVF virus. The recombinant RVF viruses described hereincan also be used as live-virus research tools, particularly thoseviruses that incorporate reporter genes, for instance a fluorescentprotein such as GFP. For example, these viruses can be used forhigh-throughput screening of antiviral compounds in vitro.

Efforts to prevent RVF virus infection via vaccination began shortlyafter the first isolation of the virus in 1931 (Findlay and Daubney,Lancet ii:1350-1351, 1931). These earliest vaccines (MacKenzie, J.Pathol. Bacteriol. 40:65-73, 1935) and several that followed, includingthe currently available TSI-GSD-200 preparation, relied on formalininactivation of live wild-type virus (Pittman et al., Vaccine18:181-189, 1999; Randall et al., J. Immunol. 89:660-671, 1962). Whilecapable of eliciting protective immune responses among livestock andhumans, these inactivated vaccines typically require a series of 2 or 3initial inoculations, followed by regular booster vaccinations toachieve and maintain protection (Pittman et al., Vaccine 18:181-189,1999; Swanepoel et al., “Rift Valley fever” in Infectious Diseases oflivestock with special reference to South Africa, pages 688-717, Oxforduniversity Press, Cape Town). However, multiple dosing and annualvaccination regimens are logistically difficult to implement andexpensive to maintain, and thus are of limited practical value inresource-poor settings, especially in regard to control of RVF virusinfection in livestock in enzootic settings. In addition, there havebeen problems in the past with quality control and “inactivated”vaccines causing disease.

In an effort to eliminate the necessity of booster inoculations, severallive-attenuated vaccine candidates were developed for RVF virus withsome, such as the Smithburn neurotropic strain, being employed inAfrica. These vaccine candidates have relied upon the randomintroduction of attenuating mutations via serial passage in sucklingmouse brain or tissue culture, in vitro passage in the presence ofchemical mutagens, such as 5-fluorouracil, or as naturally occurringvirus isolates (such as the Smithburn neurotropic strain, the Kenyan-IB8strains, MP-12, or the Clone 13 isolate) (Caplen et al., J. Gen. Virol.66:2271-2277, 1985; Coackley, J. Pathol. Bacteriol. 89:123-131, 1965;Moussa et al., Am. J. Trop. Med. Hyg. 35:660-662, 1986; Muller et al.,Am J. Trop. Med. Hyg. 53:405-411, 1995; Rossi and Turell, J. Gen. Virol.69:817-823, 1988; Smithburn, Br. J. Exp. Pathol. 30:1-16, 1949).

Due to the technical limitations of these procedures, and the lack ofcomplete genome sequence for many of the historically derived RVF virusvaccines, the exact underlying molecular mechanisms of attenuation formany of these live-attenuated RVF virus vaccines is either unknown(Smithburn neurotropic strain or Kenyan-IB8) or reliant on thecombinatorial effects of multiple nucleotide or amino acid substitutions(MP-12) (Saluzzo and Smith, Vaccine 8:369-375, 1990; Takehara et al.,Virology 169:452-457, 1989). Experimental and field experience withexisting live-attenuated RVF virus vaccines demonstrated that in certaininstances these vaccines retain the ability to cause teratogeniceffects, abortion, and neural pathology in livestock or animal models.Thus, widespread use of these live-attenuated vaccines is problematic,especially in non-endemic areas, or during inter-epizootic/epidemicperiods (Hunter et al., Onderstepoort J. Vet. Res. 69:95-98, 2002;Morrill et al., Am. J. Vet. Res. 58:1104-1109, 1997; Morrill et al., Am.J. Vet. Res. 58:1110-1114, 1997; Morrill and Peters, Vaccine21:2994-3002, 2003).

While useful in many situations, several distinct disadvantages existamong live attenuated RNA virus vaccines prepared by the traditionaltechniques discussed above. Live-attenuated vaccines reliant on singleor multiple nucleotide substitutions are at increased risk for reversionto virulent phenotypes due to the inherently high rate of viral RNApolymerase errors. The loss of attenuation via this mechanism amonglivestock and human live vaccines has been documented (Berkhout et al.,J. Virol. 73:1138-1145, 1999; Catelli et al., Vaccine 24:6476-6482,2006; Halstead et al., Am. J. Trop. Med. Hyg. 33:672-678, 1984; Hopkinsand Yoder, Avian Dis. 30:221-223, 1986; Rahimi et al., J. Clin. Virol.39:304-307, 2007).

The potential for a similar reversion event among live RVF virusvaccines dependent on attenuating nucleotide mutation was illustrated byrecent genomic analyses of RVF virus that revealed an overall molecularevolution rate (˜2.5×10⁻⁴ nucleotide substitutions/site/year) similar toother single-stranded RNA viruses (Bird et al., J. Virol. 81:2805-2816,2007). Due to error-prone polymerases, live-attenuated RNA virusvaccines prepared by multiple serial passage techniques involved invirus attenuation inherently consist of a complex mixture of genomicmicro-variants. In contrast, the origin of reverse genetics derivedvirus vaccine candidates is advantageous in that vaccine stocks can begenerated directly or following limited amplification steps fromprecisely defined DNA plasmids. This approach allows for the simpleproduction of virus vaccines following good manufacturing processes(GMP) with higher levels of genetic homogeneity.

Another significant drawback of all previously generated live-attenuatedRVF virus vaccines is that they do not allow for differentiation ofnaturally infected from vaccinated animals (DIVA). This ability isimportant to augment efforts to contain an accidental or intentionalrelease of wild-type RVF virus in previously unaffected areas(Henderson, Biologicals 33:203-209, 2005). As a high consequencepathogen, RVF virus has been classified as a category A Select Agent asdefined by the United States Department of Health and Human Services andthe United States Department of Agriculture (USDA), and is listed as ahigh consequence agent with potential for international spread (List A)by the Office International des Epizooties (OIE) (Le May et al., Cell116:541-550, 2004) of the World Organization for Animal Health (WOAH),thus greatly increasing the consequences for international livestocktrade following the introduction of RVF virus into previously unaffectedcountries or epizootics in enzootic areas (USDA, 7 CFR Part 331 and 9CFR Part 121, Federal Register RIN 0579-AB47:13241-13292, 2005).Currently, OIE regulations require surveillance and absence of RVF virusactivity for 2 years following an outbreak before resumption of diseasefree status and the subsequent easing of import/export traderestrictions (International Office of Epizootics, Terrestrial AnimalHealth Code, XI:2.2.14.1, 2007). The use of any current commerciallyavailable livestock vaccines does not permit the differentiation ofvaccinated from naturally infected livestock, thus contraindicating theuse of prophylactic vaccination in countries wishing to retain diseasefree status, or in those with ongoing/endemic RVF virus activity.

Thus, disclosed herein are infectious recombinant RVF viruses, generatedusing reverse genetics, containing either complete deletions of majorvirus virulence factors, NSs (rZH501-ΔNSs:GFP) or NSs and NSm(rZH501-ΔNSs:GFP-ΔNSm), which confer attenuated phenotypes in vivo, andwhich allow for the serologic differentiation of naturally infected andvaccinated animals by presence/absence of anti-RVF NP/anti-RVF NSsantibodies. As described herein, in vivo testing of these recombinantRVF (rRVF) viruses demonstrated that they were highly immunogenic andefficacious in the prevention of severe RVF virus disease and lethality(FIG. 2 and FIG. 4). In an initial pilot study, animals developed highend-point titers (≧1:400) of total anti-RVF virus IgG by day 21post-vaccination that were significantly higher than sham inoculatedcontrols (p-value <0.05, Table 1). At no observed time pointpost-vaccination did any animal develop disease symptoms or vaccinevirus induced viremia (FIG. 4).

Additional testing in a larger follow-up study confirmed these resultswith the majority of animals generating robust total anti-RVF IgGresponses with typical titers ≧1:6400 by day 26 post-vaccination. Aswith the pilot study, no vaccine virus induced viremia was detected. Theimmunologic response generated in the ΔNSs/ΔNSm virus vaccinated animalswas significantly higher than controls (p-value <0.05) and wassufficient to confer complete protection from both clinical illness andlethality in 100% of vaccinated animals given a known lethal challengedose of wild-type RVF virus (FIG. 4).

Direct comparisons of the level of protective immunity (PRNT₅₀ or totalIgG) titers with previous studies utilizing other RVF virus vaccines aredifficult due to vaccine used and species level differences in immunity.However, earlier studies utilizing the 3 dose regimen (day 0, 7 and 28)of inactivated TSI-GSD-200 vaccine in the WF rat model demonstratedprotective efficacy against virulent virus challenge at PRNT₈₀titers >1:40 (Anderson et al., Vaccine 9:710-714, 1991). Later, a largeretrospective analysis of human volunteers (n=598), receiving the samerecommended 3-dose regimen of this inactivated vaccine were found todevelop mean PRNT₈₀ titers of 1:237 (Pittman et al., Vaccine 18:181-189,1999). Additionally, a large study of the pathogenesis andneurovirulence of the live-attenuated MP-12 vaccine in rhesus macaquesdemonstrated PRNT₈₀ titers among vaccinated animals of ≧1:640 (Morrilland Peters, Vaccine 21:2994-3002, 2003). As described herein,inoculation with a single dose of the recombinant RVF viruses resultedin mean PRNT₅₀ titers ranging from 1:640 to 1:7040, indicating that thelevel of neutralizing antibody is similar to or greater than thatdemonstrated in earlier RVF vaccine studies using multiple doses inanimal model systems or among human volunteers.

Thus, the enhanced safety, attenuation, and reduced possibility ofreversion to full virulence (via either RVF virus polymerase nucleotidesubstitution or gene segment reassortment with field-strains) affordedby the double genetic deletions of the entire RVF virus NSs and NSmgenes, does not diminish overall vaccine efficacy. A high level ofprotective immunity was induced by a single dose of the rRVF virusesdisclosed herein, with 37 of 40 total vaccinated animals developing apotentially sterilizing immunity as determined by the lack of anydetectable post-challenge viremia (FIG. 4).

As described herein, animals immunized with either recombinant RVF virusdo not have detectable anti-NSs antibodies. Thus, given the high-levelanti-NSs antibody in survivor control animals, DIVA will be possibleamong animals immunized with these candidate vaccines based on thepresence/absence of anti-NSs antibody (FIGS. 3A-C). Anti-NSs antibodieshave also been detected in the serum of naturally infected convalescentlivestock obtained during the outbreak in Saudi Arabia in 2000 (FIG.3D), and in humans. Therefore, the use of these recombinant RVF viruses,combined with the further development of rapid ELISA or solidmatrix-based differential detection assays for anti-NP/anti-NSsantibodies, can provide a robust DIVA field screening tool.

In addition, the recombinant RVF viruses described herein routinely grewto high titers in tissue culture and provided protective immunityfollowing a single injection, thus likely reducing the overall economiccost of production, and potentially eliminating the need for resourceintensive follow-up booster inoculations. Additionally, the preciselydefined attenuating deletions and use of cDNA technology eliminates thepotential risk of reversion to, or contamination from, virulentwild-type virus inherent in serial passaged or inactivated vaccinepreparations, and may ease the federal/national regulatory approvalprocess. While the recombinant RVF viruses can be targeted towardsveterinary medical use, and thus indirectly the prevention of human RVFdisease, the candidate vaccines can also provide effective prophylacticprotection for humans, such as those in high risk occupational settings,or in recognized risk groups following natural or intentionalintroduction of RVF virus into previously unaffected areas.

VII. Administration of Recombinant RVF Virus for Vaccination

Recombinant RVF viruses, or immunogenic compositions thereof, can beadministered to a subject by any of the routes normally used forintroducing recombinant virus into a subject. Methods of administrationinclude, but are not limited to, intradermal, intramuscular,intraperitoneal, parenteral, intravenous, subcutaneous, vaginal, rectal,intranasal, inhalation or oral. Parenteral administration, such assubcutaneous, intravenous or intramuscular administration, is generallyachieved by injection. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution or suspension in liquid prior to injection, or asemulsions. Injection solutions and suspensions can be prepared fromsterile powders, granules, and tablets of the kind previously described.Administration can be systemic or local.

Immunogenic compositions are administered in any suitable manner, suchas with pharmaceutically acceptable carriers. Pharmaceuticallyacceptable carriers are determined in part by the particular compositionbeing administered, as well as by the particular method used toadminister the composition. Accordingly, there is a wide variety ofsuitable formulations of pharmaceutical compositions of the presentdisclosure.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Some of the compositions may potentially be administered as apharmaceutically acceptable acid- or base-addition salt, formed byreaction with inorganic acids such as hydrochloric acid, hydrobromicacid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, andphosphoric acid, and organic acids such as formic acid, acetic acid,propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid,malonic acid, succinic acid, maleic acid, and fumaric acid, or byreaction with an inorganic base such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and organic bases such as mono-, di-,trialkyl and aryl amines and substituted ethanolamines.

Administration can be accomplished by single or multiple doses. The doseadministered to a subject in the context of the present disclosureshould be sufficient to induce a beneficial therapeutic response in asubject over time, or to inhibit or prevent RVF virus infection. Thedose required will vary from subject to subject depending on thespecies, age, weight and general condition of the subject, the severityof the infection being treated, the particular immunogenic compositionbeing used and its mode of administration. An appropriate dose can bedetermined by one of ordinary skill in the art using only routineexperimentation.

Provided herein are pharmaceutical compositions (also referred to asimmunogenic compositions) which include a therapeutically effectiveamount of the recombinant RVF viruses alone or in combination with apharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers include, but are not limited to, saline, buffered saline,dextrose, water, glycerol, ethanol, and combinations thereof. Thecarrier and composition can be sterile, and the formulation suits themode of administration. The composition can also contain minor amountsof wetting or emulsifying agents, or pH buffering agents. Thecomposition can be a liquid solution, suspension, emulsion, tablet,pill, capsule, sustained release formulation, or powder. The compositioncan be formulated as a suppository, with traditional binders andcarriers such as triglycerides. Oral formulations can include standardcarriers such as pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, and magnesiumcarbonate. Any of the common pharmaceutical carriers, such as sterilesaline solution or sesame oil, can be used. The medium can also containconventional pharmaceutical adjunct materials such as, for example,pharmaceutically acceptable salts to adjust the osmotic pressure,buffers, preservatives and the like. Other media that can be used withthe compositions and methods provided herein are normal saline andsesame oil.

The recombinant RVF viruses described herein can be administered aloneor in combination with other therapeutic agents to enhance antigenicity.For example, the recombinant viruses can be administered with anadjuvant, such as Freund incomplete adjuvant or Freund's completeadjuvant.

Optionally, one or more cytokines, such as IL-2, IL-6, IL-12, RANTES,GM-CSF, TNF-α, or IFN-γ, one or more growth factors, such as GM-CSF orG-CSF; one or more molecules such as OX-40L or 41 BBL, or combinationsof these molecules, can be used as biological adjuvants (see, forexample, Salgaller et al., 1998, J. Surg. Oncol. 68(2):122-38; Lotze etal., 2000, Cancer J. Sci. Am. 6(Suppl 1):S61-6; Cao et al., 1998, StemCells 16(Suppl 1):251-60; Kuiper et al., 2000, Adv. Exp. Med. Biol.465:381-90). These molecules can be administered systemically (orlocally) to the host.

A number of means for inducing cellular responses, both in vitro and invivo, are known. Lipids have been identified as agents capable ofassisting in priming CTL in vivo against various antigens. For example,as described in U.S. Pat. No. 5,662,907, palmitic acid residues can beattached to the alpha and epsilon amino groups of a lysine residue andthen linked (for example, via one or more linking residues, such asglycine, glycine-glycine, serine, serine-serine, or the like) to animmunogenic peptide. The lipidated peptide can then be injected directlyin a micellar form, incorporated in a liposome, or emulsified in anadjuvant. As another example, E. coli lipoproteins, such astripalmitoyl-S-glycerylcysteinlyseryl-serine can be used to prime tumorspecific CTL when covalently attached to an appropriate peptide (see,Deres et al., Nature 342:561, 1989). Further, as the induction ofneutralizing antibodies can also be primed with the same moleculeconjugated to a peptide which displays an appropriate epitope, twocompositions can be combined to elicit both humoral and cell-mediatedresponses where that is deemed desirable.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the disclosure to the particular features or embodimentsdescribed.

EXAMPLES Example 1 Optimized Reverse Genetics System for Generation ofRecombinant RVF Virus

This example describes the development of an improved reverse geneticssystem for generation of recombinant RVF viruses. The recombinant RVFviruses described herein are generated using an optimized reversegenetics system capable of rapidly generating wild-type and mutantviruses (Bird et al., Virology 362:10-15, 2007, herein incorporated byreference). The RVF virus reverse genetics system is a T7 RNApolymerase-driven plasmid-based genetic system that requires only threeplasmids. Each plasmid individually expresses an anti-genomic copy ofeither the S, M or L segment. In this specific example, the S, M and Lsegments are derived from the genome of the virulent Egyptian RVF virusisolate ZH501. Nucleotide sequences of ZH501 S, M and L segments areprovided herein as SEQ ID NOs: 1-3, respectively.

Rescue of recombinant viruses is accomplished by simultaneoustransfection of the three anti-genomic sense plasmids into cells stablyexpressing T7 polymerase (for example, BSR-T7/5 cells). The genomesegments of each plasmid are flanked by a T7 promoter, which enablesgeneration of the primary RNA transcript, and the hepatitis delta virusribozyme, which removes extraneous nucleotides from the 3′ end of theprimary transcriptional products. The T7 RNA polymerase generatedtranscripts are identical copies of the RVF-encoding plasmids, with theexception of an extra G nucleotide on the 5′ end derived from the T7promoter.

A previously reported method of producing recombinant RVF virus requiredthe use of five expression plasmids (Gerrard et al., Virology359:459-465, 2007). In addition to expression plasmids encoding RVFvirus S, M and L segments, this system included plasmids encoding RVFvirus N protein and RNA-dependent RNA polymerase (L protein). Northernblot and 3′RACE revealed that the S plasmid used in that system producedlow levels of full length S-segment anti-genomic replication products.To produce a more efficient S segment plasmid, the full length S RNAfrom ZH501 was re-cloned into a version of the plasmid vector that wasmodified to remove the multiple cloning site. Within this backbonecontext, levels of N protein expression were significantly increased,allowing for subsequent rescue of recombinant virus without the need fora support plasmid encoding the N protein.

In addition, the optimized system described herein includes an M segmentclone in which two non-synonymous mutations were corrected to match thewild-type sequence, allowing for exact in vivo comparisons betweenrecombinant and wild-type viruses. These corrections were made in boththe full-length M segment and the ΔNSm plasmid by site directedmutagenesis and restriction fragment exchange. These changes includedremoval of the XhoI restriction site. After incorporating these changesinto the reverse genetics system, virus rescue was accomplished bysimultaneous transfection of only the three anti-genomic sense plasmids(either wild-type S, M and L segments, or deletion mutants thereof). Thenucleotide sequences of exemplary wild-type S, M and L plasmids, andplasmids comprising deletions in the S and M segments, are providedherein as SEQ ID NOs: 11-15. Maps of each of these plasmids are shown inFIGS. 5-9.

After complete lysis of transfected cells, cell supernatants containingrescued viruses were clarified, diluted and virus passaged twice on VeroE6 cells. All recombinant viruses were found to grow similarly towild-type ZH501 in Vero E6 cell culture. To confirm the exact molecularidentity of all viruses used in this Example, complete genome sequencewas obtained following previously described techniques (Bird et al., J.Virol. 81(6):2805-2816, 2007, herein incorporated by reference).

A total of 11 separate rescue attempts using a variety of plasmidconcentrations ranging from 0.5 μg to 4 μg resulted in 100% recombinantvirus rescue efficiency using only the three expression plasmids.

Example 2 Generation and Characterization of Recombinant RVF (rRVF)Viruses

This example describes the generation of recombinant RVF virusescomprising deletions in the ORF of NSs and/or NSm, which play a role inviral virulence. This example further describes a recombinant RVF viruscomprising a reporter gene.

RVF Virus and Biosafety

All work with infectious RVF virus (wild-type or recombinant) wasconducted within the Centers for Disease Control (CDC) bio-safety level4 (BSL-4) laboratory. Low passage (FRhL+2, Vero E6+2) working stocks ofwild-type strain ZH501, isolated originally from a fatal Egyptian humancase in 1977, were used as challenge virus, and were prepared by passageon Vero E6 cell monolayers. The complete genome sequence of the S, M andL segments of the wild-type RVF virus strain ZH501 has been depositedunder Genbank Accession Nos. DQ380149 (SEQ ID NO: 1), DQ380200 (SEQ IDNO: 2) and DQ375406 (SEQ ID NO: 3), respectively.

Construction of ΔNSs:GFP Deletion/Replacement Plasmid

To generate plasmids containing a complete deletion of the NSs ORF,replaced by the eGFP ORF (Towner et al., Virology 332(1):20-27, 2005,incorporate herein by reference), the eGFP ORF was amplified by PCR withforward and reverse primers containing the KpnI (GGTACC) and BglII(AGATCT) restriction sites, respectively. Utilizing the full-length RVFS-segment plasmid (pRVS; SEQ ID NO: 11), a strand specific PCR wasconducted with primers designed to contain the KpnI and BglIIrestriction sites annealing immediately upstream to the NSs START codon,and immediately down-stream of the NSs STOP codon. Primer sequences areshown below in Table 1. Restriction site sequences are indicated byitalics.

TABLE 1 Primer sequences for replacement of NSs with eGFP SEQ ID PrimerSequence NO: RVS - 35/KpnI aaaaaaGGTACCGATATACTTGATA 4 AGCACTAG RVS +827/BglII aaaaaaAGATCTGATTAGAGGTTAA 5 GGCTG eGFP + 1/KpnIaaaaaaGGTACCATGGTGAGCAAGG 6 GCGAGGAG eGFP - 720/BglIIaaaaaaAGATCTTTACTTGTACAGC 7 TCGTCCATG

The resulting PCR fragment contained a complete deletion of the NSs ORFflanked by KpnI and BglII restrictions sites. The eGFP and RVF-S plasmidamplicons were gel purified and ligated following standard molecularbiology techniques. The resulting plasmid (pRVS-GFPΔNSs; SEQ ID NO: 14)contained a complete in-frame replacement of the NSs ORF by eGFP.

Construction of Plasmids Containing a NSs-eGFP Fusion Protein

In a second set of plasmid constructions, rRVF viruses were generatedwhich contained the complete full-length genome and an insertion of theeGFP ORF. Two constructions were made containing in-frame fusions of thec-terminus of NSs with the eGFP ORF, separated by amino acid linkermoieties of varying lengths. In the first construction, the full-lengthRVF-S segment plasmid backbone was modified using site directedmutagenesis PCR (QuickChange, Stratagene) with overlapping primers(shown in Table 2 below) containing a deletion/replacement of the NSsSTOP codon with a linker section containing nucleotides encoding threealanine residues and the KpnI restriction site. Following this, boththis amplicon and the pRVS-GFPΔNSs plasmid were digested with KpnI. Theresulting restriction enzyme fragments were gel purified and ligated togenerate the final construction pRVFS-NSs(A₃)eGFP. A second NSs-eGFPfusion peptide construction was created to increase the length of theeGFP linker moiety and eliminate the KpnI restriction site contained inthe previous fusion peptide construction. To accomplish this, sitedirected mutagenesis (QuickChange, Stratagene) was employed startingwith the pRVFS-NSs(A₃)eGFP backbone and overlapping primers containingthe remote cutter restriction enzyme BsmBI and nucleotides encoding tenalanine residues (shown in Table 2 below).

TABLE 2 Primer sequences for construction of NSs-eGFP fusion proteinsSEQ ID Primer Sequence NO: RVS - 829rev/ aaaaaaGGTACCTGCTGCTGC  8 KpnIATCAACCTCAACAAATCCATC RVS + 827/ aaaaaaAGATCTGATTAGAGG  5 BglIITTAAGGCTG NS sGFP + 10Ala/ aaaaaaCGTCTCaGCAGCAGC  9 FwdAGCAGCAATGGTGAGCAAGGG   CGAGGAG NS sGFP + 10Ala/ aaaaaaCGTCTCaCTGCTGCT10 Rev GCTGCTGCTGCATCAACCTCA ACAAATCCATC

Following PCR amplification, restriction enzyme digestion andre-ligation, the resulting construction contained a perfect in-framefusion of the C-terminus of NSs and eGFP ORF in the context of thecomplete full-length RVF S segment genome, preserving the spacing andnucleotide sequence of both the NP and NSs transcription terminationsignals.

Generation and In Vitro Testing of Recombinant RVF Viruses

In all cases, rescue of recombinant viruses was accomplished using cDNAplasmids encoding the virulent RVF virus ZH501 strain. The basic designand construction of the full-length plasmids containing inserts of thecomplete S segment (pRVS; SEQ ID NO: 11), M segment (pRVM; SEQ ID NO:12), and L segment (pRVL; SEQ ID NO: 13), and a plasmid containing adeletion of the NSm gene (pRVMΔNSm; SEQ ID NO: 15), have been describedpreviously (Bird et al., Virology 362:10-15, 2007; Gerrard et al.,Virology 359:459-465, 2007, each of which is herein incorporated byreference). Maps of the full-length S, M and L plasmids, and plasmidscontaining NSs and NSm deletions, are shown in FIGS. 5-9.

Anti-genomic sense plasmids representing the three genomic segments weretransfected in 1 μg quantities with LT-1 (Mirus) at a ratio of 6:1 andtransferred onto sub-confluent (approximately 60-70% confluent)monolayers of BSR-T7/5 cells stably expressing T7 polymerase (Buchholzet al., J. Virol. 73(1):251-259, 1999, herein incorporated byreference). Four or five days post transfection, the cell supernatantwas clarified by low speed centrifugation and passaged twice onconfluent monolayers of Vero E6 cells. After passage and prior to use insubsequent experiments, the complete genome sequence of each rescuedrecombinant virus was confirmed by previously described techniques (Birdet al., J. Virol. 81:2805-2816, 2007, herein incorporated by reference).

Infected Live Cell (Direct) or Fixed Cell (Indirect) FluorescentAntibody Detection of RVF NSs and eGFP Proteins

Vero E6 cells were seeded on glass coverslips and infected at amultiplicity of infection (MOI) of approximately 1.0 with eitherrZH501-ΔNSs:GFP, rZH501-ΔNSs:GFP-ΔNSm, rZH501-NSs(Ala)₃GFP orrZH501-NSs(Ala)₁₀GFP. At 24 hours post infection, cells were directlyvisualized by inverted ultraviolet microscopy (live cell) or were fixedin 10% formalin overnight. Following fixation, infected cells weregamma-irradiated (5.0×10⁶ RAD) to inactivate any residual virusactivity. After inactivation, cells were permeabilized (Triton X-1000.01%) and incubated with monoclonal antibodies specific for either RVFNSs or eGFP protein following standard techniques.

Results

Rescue of all recombinant viruses used in this study was accomplished bytransfection of three anti-genomic sense plasmids, each representing oneof the three virus RNA segments, without the requirement of supportingexpression plasmids encoding virus structural proteins. Multiple rRVFviruses were generated containing an insertion of the reporter moleculeeGFP into the virus S segment (FIG. 1A). Among these, two rRVF viruseswere rescued containing an in-frame fusion of the C-terminus of the NSsprotein with the N-terminus of eGFP, separated by a peptide linker ofeither three or ten alanine residues (rZH501-NSs(Ala)₃GFP andrZH501-NSs(Ala)₁₀GFP). Two rRVF viruses were created containingdeletions of either the NSs alone or NSs/NSm genes in combination(rZH501-ΔNSs:GFP and rZH501-ΔNSs:GFP-ΔNSm). In both of these viruses,the NSs gene was replaced by the reporter molecule eGFP, preserving thenative S segment ambisense RNA orientation. Both rRVF viruses wererescued upon the first attempt and grew to high titers routinelyexceeding 1.0×10⁶ PFU/mL in Vero E6 cell culture resulting in completemonolayer lysis.

Following passage on Vero E6 cells, cytoplasmic GFP signal was firstobserved approximately 10-12 hours post infection and appeared to spreadrapidly throughout the cell monolayer prior to the first signs ofextensive CPE and plaque formation (FIG. 1B). Recombinant viruscontaining NSs-GFP fusion peptides (rZH501-NSs(Ala)₃GFP andrZH501-NSs(Ala)₁₀GFP) were also rescued on the first attempt but werefound to grow to slightly lower titers (about 5.0×10⁵ PFU/mL). NSs-GFPfusion protein was first localized in the cytoplasm of infected cellsfollowed by perinuclear accumulation and eventual intranuclear migrationfollowed by the formation of filamentous structures by 12-18 hourspost-infection (FIGS. 1B and 1C) Stability of the eGFP reporter gene inall recombinant viruses reported here was followed for 15 serialpassages (1:100 dilution between each passage) in Vero E6 cells duringwhich time no decrease in the stability of eGFP expression was observed,with all infected cells expressing robust amounts of eGFP proteinsimilar to that seen in early passages.

Example 3 In Vivo Immunogenicity and Safety of rRVF Viruses

This example describes a study to determine the immunogenicity andsafety of the recombinant RVF viruses described in the examples above.

Animal Immunization and Infection

A total of 66 female Wistar-furth (WF/NSd) (Harlan) rats 6-8 weeks ofage (approximately 160 g) were used in this study. The animals werehoused in micro-isolator pans and provided food and water ad libitum.All pans were kept in HEPA filtration racks following standard barriercare techniques within the BSL-4 laboratory for the duration of theexperiment. All animals were inoculated subcutaneously (SQ) in the righthind flank with an inoculum (vaccine or challenge virus) prepared in 200μL of sterile physiologic saline. A total of eight animals that wereadministered sterile saline only served as sham inoculated animalcontrols (three in the pilot study, five in the vaccination/challengestudy). All animals were examined daily post inoculation for signs ofclinical illness, weight loss and respiratory distress. Animals foundeither in distress or moribund were immediately anesthetized usingisoflurane and then euthanized using Beuthanasia solution(Schering-Plough) following standard techniques.

Pilot In Vivo Immunogenicity and Safety Study

A total of 18 rats were administered 1.0×10³ PFU SQ of either therZH501-ΔNSs:GFP (nine animals) or rZH501-ΔNSs:GFP-ΔNSm (nine animals)with three animals serving as sham inoculated controls. Followingvaccination, a small (approximately 25 μL) sample of whole blood wasobtained via the tail vein on days 1-4 to detect vaccine virus inducedviremia. This whole blood sample was placed directly into 2×non-cellular lysis buffer (Applied Biosystems) for decontamination andtransfer to a BSL-2 laboratory following standard protocols (Towner etal., J. Infect. Dis. S2002-S212, 2007, incorporated herein by reference)for subsequent RNA extraction and RVF specific q-RT-PCR. At day 21post-vaccination, all animals were anesthetized using isoflurane, serumsamples were collected for determination of total anti-RVF IgG titers,and the animals were euthanized using Beuthanasia solution(Schering-Plough).

Anti-RVF Virus Total IgG ELISA

Determination of anti-RVF IgG titers from vaccinated and control ratswas performed essentially as described by Madani et al. (Clin. Infect.Dis. 37:1084-1092, 2003) with the following modifications necessary forrat specimens. Standard 96-well microtiter plates were coated overnightat 4° C. with 100 μL of gamma-irradiated RVF infected Vero E6 celllysate diluted 1:2000 in (0.01M PBS, pH 7.2), or similarly dilutedgamma-irradiated uninfected Vero E6 cell lysate, to serve as adsorptioncontrols. Plates were washed 3× (PBS 0.01% Tween-20) and 100 μLduplicate samples of rat sera were diluted 1:100-1:6400 in 4-folddilutions in skim milk serum diluent and adsorbed for 1 hour at 37° C.Plates were washed 3× (PBS 0.01% Tween-20) and 100 μL of goat anti-RatIgG HRP conjugate antibody (KPL, Gaithersburg, Md.) diluted 1:2500 wasadsorbed for 1 hour at 37° C. After a 3× wash, 100 μL of ABTS substrate(KPL) was added and incubated for 30 minutes at 37° C.

Plates were read at 410 nm with an absorbance correction of 490 nm forplate imperfections. The absorbance of the 1:100, 1:400, 1:1600, and1:6400 dilutions were added and constituted a SUM_(OD) value for eachspecimen. The background adsorption of each animal serum to negativecontrol Vero E6 cells was subtracted from the calculated SUM_(OD) valueobtained from antigen positive Vero E6 cells and was recorded as anadjusted-SUM_(OD) value for each animal. Final end point dilution titerswere determined as the reciprocal of the final serum dilution yieldingan adjusted-SUM_(OD) of >0.20. A cut-off value for determining positiveversus negative samples was established as the mean sampleadjusted-SUM_(OD)+3 standard deviations obtained from the five shaminoculated control animals.

Statistical Analyses

For all calculations, the analysis program XLSTAT (AddinSoft, USA) wasutilized. Kaplan-Meier analyses were completed with log-rank andWilcoxan tests of significance with an α-level setting of 0.05. Analysesof SUM_(OD) and viremia were completed utilizing a one-way ANOVA and apost-hoc Tukey's HSD test of significance with an α-level setting of0.05.

Results

To gain a primary assessment of the relative in vivo characteristics ofthe rZH501-ΔNSs:GFP and rZH501-ΔNSs:GFP-ΔNSm recombinant viruses, groupsof nine rats were inoculated with each rRVF virus at a dose of 1.0×10³PFU (FIG. 4A). Animals were monitored daily for signs of clinicalillness and weight loss. At no time post-vaccination did any animal showsigns of clinical illness, and all experienced average daily weightchanges equivalent to sham inoculated controls of approximately 0-5 g.All vaccinated rats were bled at days 1-4 post-inoculation to determinethe titer of vaccine virus induced viremia. Using a highly sensitiveq-RT-PCR assay, no animal at any time point analyzed post-vaccinationdeveloped a detectable viremia.

All animals were euthanized at day 21 post-vaccination and anti-RVFtotal IgG antibody titers were evaluated (FIG. 2). Testing revealed thatthe SUM_(OD) (mean±SEM) for all animals vaccinated with therZH501-ΔNSs:GFP virus was 2.14±0.12, which corresponded to end pointtiters of 1:1600 in 66% (6/9) of animals, with the remaining 33% (3/9)having titers equal to 1:400. Among animals receiving therZH501-ΔNSs:GFP-ΔNSm virus, SUM_(OD) (mean±SEM) was 1.24±0.06, with 89%(8/9) developing end-point dilution titers equal to 1:400. As expected,all sham-inoculated animals (N=3) were negative for detectable levels ofanti-RVF total IgG antibody SUM_(OD) −0.08±0.06. All vaccinated animalsin the rZH501-ΔNSs:GFP and rZH501-ΔNSs:GFP-ΔNSm virus groups developedstatistically higher mean anti-RVF total IgG SUM_(OD) values comparedwith non-vaccinated controls (p-value <0.001 and p-value=0.003,respectively). Animals in the rZH501-ΔNSs:GFP virus group developedsignificantly higher mean SUM_(OD) values than animals given therZH501-ΔNSs:GFP-ΔNSm vaccine (p-value=0.004). Plaque reductionneutralization titers (PRNT₅₀) testing was completed on a subset (fouranimals) chosen randomly from each vaccine virus group with two shaminoculated animals serving as controls. The results showed mean PRNT₅₀titers of 1:1480 (rZH501-ΔNSs:GFP) and 1:280 (rZH501-ΔNSs:GFP-ΔNSm),with sham inoculated control animal titer of ≦1:10.

Example 4 Follow-Up Vaccination and Virulent Virus Challenge Study

This example describes the efficacy of recombinant RVF virusescomprising a deletion in the NSs ORF, or comprising a deletion in boththe NSs and NSm ORFs, following challenge with wild-type virus.

Vaccination and Virus Challenge

A total of 20 rats were vaccinated in two dosage groups of ten animalseach with either 1.0×10³ or 1.0×10⁴ PFU SQ of the rZH501-ΔNSs:GFP virusas described above. An additional 20 rats were inoculated in two dosagegroups of ten animals each with either 1.0×10³ or 1.0×10⁴ PFU SQ of therZH501-ΔNSs:GFP-ΔNSm virus as described above. A total of five animalsserved as sham inoculated controls. On days 2, 4 and 7 post-vaccination,a small blood sample (approximately 25 μL) was collected from the tailvein and added directly to 2× non-cellular lysis buffer (AppliedBiosystems) as described above for subsequent RNA extraction andq-RT-PCR.

At day 26 post-vaccination, all animals were briefly anesthetized usingisoflurane vapor (3.0-3.5% atmosphere) (RC² Rodent Anesthesia system,VetEquip) and a 500 μL sample of whole blood was obtained. Serum wascollected and stored at −70° C. for later determination of totalanti-RVF IgG titers, PRNT₅₀ and anti-NSs/anti-NP specific antibody. Allserum samples were surface decontaminated and inactivated bygamma-irradiation (5.0×10⁶ RAD) following standard BSL-4 safetyprotocols prior to use in a BSL-2 laboratory.

At day 28 post-vaccination, all rats (vaccinated and sham controls) werechallenged with 1.0×10³ PFU SQ of virulent wild-type RVF virus strainZH501, previously shown to result in lethal disease in Wistar-furth rats(Anderson et al., Microb. Pathog. 5:241-250, 1988; Anderson et al., Am.J. Trop. Med. Hyg. 44(5):475-80, 1991; Bird et al., Virology 362:10-15,2007, each of which is herein incorporated by reference). On days 2, 3,4 and 7 following challenge, a small blood sample was collected from thetail vein for subsequent RNA extraction and RVF specific q-RT-PCR toassess the level of viremia. At day 42 post-challenge, all animalssurviving wild-type virus infection were bled via cardiac puncture undergeneral anesthesia (isoflurane vapor, RC² Rodent Anesthesia system,VetEquip) followed by euthanasia (Beuthanasia solution,Schering-Plough).

Anti-RVF Virus Plaque Reduction and Neutralization Testing (PRNT₅₀)

The stock of RVF virus was diluted to 50 PFU in 300 μL of DMEM (1%Penicillin/Streptomycin) without FBS. Separately, aliquots of serum fromvaccinated rats or from sham inoculated controls corresponding to 21days post-vaccination (pilot study) or 26 days (challenge study) wereheat inactivated for 30 min at 56° C. After inactivation, serumdilutions of 1:10, 1:40, 1:160, 1:640, 1:2560 and 1:10240 were made inDMEM (1% Pen/Strep). Diluted rat serum (300 μL) was mixed with an equalvolume of diluted virus and incubated overnight at 4° C. The followingday, each mixture of serum+RVF virus was used to infect confluentmonolayers of Vero E6 cells in 12-well plates. After a 1 hr adsorption,the mixture was removed and a 1-2 ml nutrient agarose overlay (MEM 1×,2% FBS, 1% Pen/Strep, 1% SeaPlaque agar) was added to the monolayers.After a five-day incubation, the cells were fixed with 10% formalinovernight. Following fixation, the agarose overlay was removed and theplates were surface decontaminated and gamma-irradiated (2.0×10⁶ RAD)following standard BSL-4 safety procedures. After inactivation, the cellmonolayer was stained with 1% crystal violet in PBS and plaques wereenumerated. The calculated PRNT₅₀ titers correspond to the reciprocaltiter of the last dilution resulting in a 50% reduction in the number ofplaques when compared to controls.

RVF Virus Specific q-RT-PCR

Whole rat blood was assayed for the presence and quantity of RVFspecific virus RNA as described previously (Bird et al., J. Clin.Microbiol. 45(11):3506-13, 2007, incorporated herein by reference).Quantification of total serum RVF RNA was calculated directly viainterpolation from a standard curve generated from serial dilutions ofstock RVF virus strain ZH501 of a known titer in whole rat bloodextracted and processed in an identical manner with each experimentalreplicate q-RT-PCR run. Briefly, 25 μL of whole rat blood (either fromvaccinated/challenged animals or stock virus serial dilutions) was addedto 300 μL 2× non-cellular lysis buffer (Applied Biosystems) and totalRNA was extracted (ABI 6100 nucleic acid workstation, AppliedBiosystems.) After extraction, cDNA was generated by random hexamerpriming (High Capacity cDNA kit, Applied Biosystems) followed by RVFspecific q-PCR (Universal q-PCR master mix, Applied Biosystems). Resultsare reported as RVF PFU equivalents/mL of rat blood.

Results Immunization Phase

Following the promising findings of robust immunogenicity and in vivoattenuation in the initial pilot study, a larger study was undertakenutilizing multiple doses of each recombinant RVF virus followed byvirulent virus challenge. Groups of ten animals each were inoculatedwith either rZH501-ΔNSs:GFP or rZH501-ΔNSs:GFP-ΔNSm viruses at dosagesof 1.0×10³ or 1.0×10⁴ PFU (FIG. 4B). Whole blood samples were assayed atday 2, 4, and 7 post-vaccination for the presence of detectable viremia.As was observed in the pilot study, at no time did any animal developdetectable vaccine viremia. Additionally, no clinical illness wasobserved among any vaccinated animals, and all experienced weight gainsof approximately 1-5 g per day, similar to sham inoculated controls.

At day 26 post-vaccination, serum samples were obtained to determine thelevel of total anti-RVF IgG, PRNT₅₀, and anti-NP/anti-NSs proteinspecific antibody production prior to subsequent challenge on day 28.All animals regardless of recombinant virus type or dose, developedhigh-titered total anti-RVF IgG antibody (FIG. 2). Among rats receivingthe rZH501-ΔNSs:GFP virus, the mean SUM_(OD)±SEM was 4.10±0.12 (1.0×10³dose group) and 4.79±0.11 (1.0×10⁴ dose group), which corresponded to85% (17/20) of animals developing anti-RVF IgG end-point titers of1:6400. The remaining three animals in the rZH501-ΔNSs:GFP virus groupdeveloped end-point titers equal to 1:1600. In animals receiving therZH501-ΔNSs:GFP-ΔNSm virus, the mean SUM_(OD)±SEM was 3.94±0.12 (1.0×10³dose group) and 4.54±0.11 (1.0×10⁴ dose group), which corresponded to75% (15/20) developing anti-RVF IgG end-point titers of 1:6400, with theremaining 25% (5/20) attaining end-point titers of 1:1600. As wasobserved in the pilot study, all animals vaccinated with eitherrZH501-ΔNSs:GFP or rZH501-ΔNSs:GFP-ΔNSm viruses, regardless of dose,developed statistically significant higher mean SUM_(OD) values thansham inoculated control animals (p-values all <0.05).

Similarly, PRNT₅₀ titers were found to be elevated above sham inoculatedcontrols among animals inoculated with the rZH501-ΔNSs:GFP virus withmean titers of 1:640 and 1:7040 in the 1.0×10³ and 1.0×10⁴ dose groups,respectively. Mean PRNT₅₀ titers among animals vaccinated with therZH501-ΔNSs:GFP-ΔNSm were found to be similar with mean titers of 1:1120and 1:640 in the 1.0×10³ and 1.0×10⁴ dose groups, respectively.

Challenge Phase

On day 28 post-vaccination, all rats were challenged with a known lethaldose (1.0×10³ PFU) of virulent wild-type strain ZH501. All animals weremonitored daily for signs of clinical illness and weight loss/gain for42 days post-challenge. At no time post challenge (days 1-42) did anyrat that received prior vaccination with either the rZH501-ΔNSs:GFP orrZH501-ΔNSs:GFP-ΔNSm viruses develop clinically detectable illness(ruffled fur, hunched posture or lethargy). At approximately day 2post-challenge, a majority of animals suffered slight 1-5 g reductionsin total body weight that was regained by day 5 post-challenge.

Rat whole blood was obtained on days 2, 3, 4 and 7 post-challenge andassayed for the presence of RVF virus RNA by a highly sensitive q-RT-PCRassay (Bird et al., J. Clin. Microbiol. 45(11):3506-13, 2007,incorporated herein by reference). Following challenge, low-levelviremia was detected in a total of 3/40 vaccinated animals. Among the 20animals that were vaccinated with rZH501-ΔNSs:GFP-ΔNSm, two animalsdeveloped a peak post-challenge viremia on day 3 of 1.1×10² and 7.0×10¹PFU equivalents/mL of whole blood, respectively (FIG. 4B). In the 20animals vaccinated with rZH501-ΔNSs:GFP, one animal was detected with apeak post-challenge viremia on day 4 of 1.5×10¹ PFU equivalents/mL ofwhole blood. In all three animals, the detectable viremia was transientand was resolved by day 7 post-challenge. No other vaccinated animals(37 total) had a detectable RVF virus viremia at any time point assayedpost challenge. In sharp contrast, the five sham inoculated animals allsuffered severe to lethal clinical illness with 3/5 animals succumbingto infection by day 3 post-challenge with peak viremia titers of1.9×10⁷, 2.7×10⁷, and 3.1×10⁷ PFU equivalents/mL. Two sham-inoculatedanimals did not succumb to infection but did develop severe clinicalillness (ruffled fur, hunched back, lethargy) with a peak viremiapost-challenge of 2.7×10⁴ and 5.8×10³ PFU equivalents/mL whole blood. Asanticipated, both candidate vaccines (rZH501-ΔNSs:GFP orrZH501-ΔNSs:GFP-ΔNSm) significantly reduced post-challenge viremia(p-value <0.0001) regardless of dose. Additionally, Kaplan-Meiersurvivor analysis (log-rank and Wilcoxan tests) of survivalpost-challenge revealed a significant protective efficacy effect withboth rRVF viruses regardless of dose when compared with sham-inoculatedcontrols (p-value <0.001).

Example 5 Differentiation of Wild Type-Infected and Vaccinated Animals

This example describes how rRVF viruses lacking NSs can be used todifferentiate animals that have been infected with the rRVF virus andwild-type virus using methods to detect antibodies specific for NSs andNP.

Anti-RVF NSs and NP Differential Indirect Fluorescent Antibody (IFA)Assays

Plasmid constructions expressing only the RVF nucleoprotein (NP) ornon-structural S (NSs) proteins were generated following techniquesdescribed previously (Niwa et al., Gene 108:193-199, 1991, incorporatedherein by reference). Briefly, oligonucleotide primers were designed toanneal within the NP or NSs ORF with the addition of a SacI and BglIIrestriction site for cloning into a polymerase II-based expressionplasmid, pCAGGS. The resulting PCR amplicons were agarose gel purified,digested with SacI and BglII, and ligated between the correspondingrestriction sites of the pCAGGS vector. Prior to use, the resultingclones, pC-NP and pC-NSs, were sequenced using standard techniques.

Following confirmation of the molecular sequence, each plasmid wastransfected separately on Vero E6 cells grown on glass coverslips in 1μg quantities at a 6:1 ratio with lipofectant solution (LT-1, Mirus).Following a 48-hour incubation, transfected cells expressing either theNP or NSs protein were fixed with 10% formalin. To assess the presenceor absence of anti-NP or anti-NSs antibody, serum samples from allvaccinated and the two surviving sham inoculated control animals wereadsorbed separately for 1 hour on cells transfected with either NP orNSs. The presence or absence of anti-NP or anti-NSs adsorbed ratantibody was visualized by secondary adsorption of an AlexaFluor 594 nm(Giorgi et al., Virology 180:738-753, 1991) anti-rat total IgG(Molecular Probes/Invitrogen) antibody. Intra-nuclear localization ofNSs protein and rat antibody was confirmed by DAPI counterstaining. Toconfirm the presence of anti-RVF virus NP and NSs antibodies amongnaturally infected animals, archived serum samples collected from goatsin Saudi Arabia during the outbreak in 2000 were tested essentially asdescribed above with anti-goat specific total IgG (AlexaFluor 488 nm,FITC, Molecular Probes/Invitrogen).

Results

Serum obtained from all vaccinated and sham vaccinated animals at day 26post-vaccination was tested for the presence of anti-NP and anti-NSsspecific antibodies utilizing Vero E6 cells transfected with plasmidsexpressing either NP or NSs proteins. As a positive control, the serumobtained from the two sham vaccinated animals that survived infection,and six additional control rat serum samples (taken from animalsinoculated with a sub-lethal dose of RVF virus for validation purposes)were utilized. Control animals surviving infection developed high levelsof both anti-NP and anti-NSs antibody with strong immunostaining of bothin vitro expressed cytoplasmic NP and filamentous intra-nuclearaccumulations of NSs protein (FIG. 3A). No anti-NS or anti-NP antibodieswere detected in serum from control rats (FIG. 3C). In concordance withthe anti-RVF total IgG data, all vaccinated animals demonstrated highanti-NP specific antibody levels, and as anticipated, no vaccinatedanimal, regardless of vaccine virus or dose, developed detectableanti-NSs specific antibody (FIG. 3B). As a further step toward thedemonstration of the ability to differentiate naturally infected animalsfrom those vaccinated with the vaccines provided herein, naturallyinfected livestock were also shown to produce anti-NSs antibodiessimilar to those observed with the wild type virus infected rats (FIG.3D).

Example 6 Safety and Efficacy of Recombinant RVF Virus Vaccines inPregnant Ewes

The safety and efficacy of recombinant RVF virus vaccines can beevaluated in pregnant ewes according to procedures well known in the art(Morrill et al., Am. J. Vet. Res. 48(7):1042-1047, 1987; Baskerville etal., Res. Vet. Sci. 52:307-311, 1992, each of which is hereinincorporated by reference). By way of example, rRVF viruses areevaluated in healthy, RVF virus sero-negative, crossbred ewes atapproximately 12 weeks gestation. The ewes are housed in a biologicalcontainment facility and fed a daily ration of alfalfa hay and acommercial grain mix ration, with grass hay and water provided adlibitum. Pregnant ewes are administered recombinant RVF virussubcutaneously or intramuscularly at various doses. Mock-infected ewesserve as controls. Ewes are monitored daily for health. At various timepoints post-inoculation, blood, serum or other body fluid samples can betaken to assay RVF virus-induced viremia or anti-RVF virus antibodyproduction as described above, or other desired biological endpoints.Lambs born to inoculated ewes are allowed to remain with their dams andsuckle.

To test efficacy of the recombinant RVF viruses as vaccines, inoculatedand sham-inoculated ewes are administered wild-type RVF virus at variousdoses. Lambs can also be challenged with live virus to determine whethermaternal antibodies against recombinant RVF virus provide protectionagainst natural infection. Animals are observed daily for signs ofclinical illness, weight loss and respiratory distress. Animals that arein distress or moribund are immediately anesthetized and theneuthanized. As described above, at various time points followinginoculation, small blood samples can be taken to test for the presenceof viral RNA. Serum samples can be collected to determine anti-RVF virusantibody titers.

Example 7 Safety and Efficacy of Recombinant RVF Virus Vaccines in aRhesus Macaque Model for Human Disease

The safety and efficacy of recombinant RVF virus vaccines can beevaluated in non-human primates, such as rhesus macaques, according toprocedures well known in the art (Morrill and Peters, Vaccine21:2994-3002, 2003). By way of example, rRVF viruses are evaluated inadult (5-10 kg) and/or juvenile (1-3 kg) captive-bred rhesus monkeys(Macaca mulatta). Animals are housed in individual cages in a biosafetylevel 3 (BSL-3) biological containment facility maintained at constantroom temperature with a 12 hour light/dark photoperiod. Inoculations aregiven, and rectal temperatures and venous blood samples are taken whilethe animals are under ketamine-xylazine anesthesia.

Sero-negative monkeys are inoculated intravenously or intramuscularlywith various doses of recombinant RVF viruses. Mock-inoculated animalsserve as controls. The animals are monitored daily for clinical signs ofillness, including weakness, paralysis or any alteration of physicalcondition. At various time points post-inoculation, blood, serum orother body fluid samples can be taken to assay RVF virus-inducedviremia, anti-RVF virus antibody production, or other desired biologicalendpoints (for example, white blood cell count, red blood cell count,hematocrit, platelet count, AST and ALT). Moribund monkeys areeuthanized and necropsied.

To test efficacy of the recombinant RVF viruses as vaccines, inoculatedand sham-inoculated monkeys are administered wild-type RVF virus atvarious doses. Animals are observed daily for signs of clinical illness,weight loss and respiratory distress. Animals that are in distress ormoribund are immediately anesthetized and then euthanized. As describedabove, at various time points following inoculation, small blood samplescan be taken to test for the presence of viral RNA. Serum samples can becollected to determine anti-RVF virus antibody titers.

Example 8 Vaccination of Human Subjects with Recombinant RVF Virus

The safety and efficacy of recombinant RVF virus vaccines can beevaluated in human volunteers according to procedures well known in theart (Pittman et al., Vaccine 18:181-189, 2000, herein incorporated byreference). Typically, human volunteers are selected from those havingoccupations that put them at risk of infection with RVF virus, such asveterinarians in endemic areas. All volunteers are screened to ensurethey are in good health. Informed consent is obtained from eachvolunteer prior to vaccination.

In this example, human volunteers are injected with candidate rRVFvaccine subcutaneously at an appropriate dose. The appropriate dose isthe dose approved by the FDA, and can be determined from suitable animalstudies conducted prior to human vaccination trials. Other routes ofadministration are possible, including intramuscular and intravenous.The vaccine can be administered as a single dose, or given in multipledoses, such as two, three or four doses. When administered in multipledoses, the booster doses can be administered at various time intervals,such as months to years. Serum samples can be obtained to determineneutralizing antibody titers and identify responder and non-respondersto the vaccine.

Vaccinated volunteers are encouraged to return and report local orsystemic reactions. Local reactions are assessed by grading pain andtenderness at the site of inoculation and/or axillary lymph nodes andmeasuring erythema and induration at the site. Systemic reactionparameters include fever, chills, headache, malaise, myalgia,arthralgia, sore throat, gastric upset, dizziness, photophobia and skinrash. Additional laboratory samples, including complete blood cellcount, chemistry profile, β-HCG (in females), urinalysis, blood samplesfor viremia titrations, and oropharyngeal washing for viral isolatedculture can be obtained. Using serum samples obtained from vaccinatedindividuals, plaque-reduction neutralization tests can be performed todetermine how robust the immune response was to the particular rRVFvirus. Vaccinated volunteers are also screened for the development ofRVF virus infection.

This disclosure provides recombinant RVF viruses comprising deletions invirus virulence genes. The disclosure further provides methods ofimmunizing subjects at risk of infection with RVF virus with therecombinant viruses. It will be apparent that the precise details of themethods described may be varied or modified without departing from thespirit of the described disclosure. We claim all such modifications andvariations that fall within the scope and spirit of the claims below.

1. A method of differentiating vaccination with a recombinant RiftValley fever (RVF) virus from a natural RVF virus infection in asubject, wherein the genome of the recombinant RVF virus comprises (i) afull-length L segment; (ii) a full-length M segment or an M segmentcomprising a complete deletion of the NSm open reading frame (ORF); and(iii) an S segment comprising a complete deletion of the NSs ORF, themethod comprising: obtaining a body fluid sample from the subject; anddetecting the presence of anti-NP antibodies and the absence of anti-NSsantibodies in the sample, thereby determining that the subject wasvaccinated with the recombinant RVF virus; or detecting the presence ofboth anti-NP and anti-NSs antibodies in the sample, thereby determiningthat the subject was naturally infected with RVF virus.
 2. The method ofclaim 1, wherein the body fluid sample is blood or serum.
 3. The methodof claim 1, wherein detecting the presence or absence of anti-NP oranti-NSs antibodies comprises an IFA assay, an ELISA assay or a solidmatrix-based differential detection assay.
 4. The method of claim 1,wherein the subject is livestock.
 5. The method of claim 4, wherein thelivestock is cattle or sheep.
 6. The method of claim 1, wherein thesubject is human.
 7. A method of differentiating vaccination with arecombinant Rift Valley fever (RVF) virus from a natural RVF virusinfection in a subject, wherein the genome of the recombinant RVF viruscomprises (i) a full-length L segment; (ii) an M segment comprising acomplete deletion of the NSm open reading frame (ORF); and (iii) an Ssegment comprising a complete deletion of the NSs ORF, the methodcomprising: obtaining a body fluid sample from the subject; anddetecting the presence of anti-NP antibodies and the absence of anti-NSmantibodies in the sample, thereby determining that the subject wasvaccinated with the recombinant RVF virus; or detecting the presence ofboth anti-NP and anti-NSm antibodies in the sample, thereby determiningthat the subject was naturally infected with RVF virus.
 8. The method ofclaim 7, wherein the body fluid sample is blood or serum.
 9. The methodof claim 7, wherein detecting the presence or absence of anti-NP oranti-NSm antibodies comprises an indirect fluorescent antibody (IFA)assay, an ELISA assay or a solid matrix-based differential detectionassay.
 10. The method of claim 7, wherein the subject is livestock. 11.The method of claim 10, wherein the livestock is cattle or sheep. 12.The method of claim 7, wherein the subject is human.
 13. The method ofclaim 7, further comprising administering to the subject the recombinantRVF virus.
 14. The method of claim 1, further comprising administeringto the subject the recombinant RVF virus.